Phosphatase and protease inhibitors

RIPA buffer supplemented with phosphatase and protease inhibitors (Beyotime Biotechnology, China) was used for heart tissues homogenization. Total protein extract (30–50 µg) was loaded to 8–12% SDS-PAGE, transferred onto PVDF membranes, blocked by non-fat dry milk (5%), and then stained with designated primary antibodies: angiotensin II receptor type 1 (AGTR1; Proteintech, 1:1000), cleaved-caspase-3 (Cell Signaling Technology, 1:1000), angiotensin-converting enzyme (ACE; Proteintech, 1:1000), Bcl-2 (Proteintech, 1:1000), Bad (Proteintech, 1:1000), and β-actin (Proteintech, 1:2000) at 4 °C for 24 h. Subsequently, the membranes were rinsed with TBST three times and incubated with goat anti-mouse (Proteintech, 1:1000) or goat anti-rabbit IgG (Proteintech, 1:1000) secondary antibody at ambient temperature for 60 min. The fluorescence signal was visualized by the ChemiDoc™ XRS + imaging system (Bio-Rad, USA) and quantified using Image J (v5.0; National Institutes of Health, USA).

Tissues and cells were lysed using RIPA buffer (Cat. No. P0013B, Beyotime, China) supplemented with phosphatase and protease inhibitors (Cat. No. P1005, Beyotime, China). Proteins were quantified using a BCA kit (Cat. No. P0010, Beyotime, China) and then separated by SDS-PAGE (Cat. No. PG113, Epizyme Biomedical Technology Co., China) before being transferred to PVDF membranes (Millipore, USA). These membranes were incubated with primary antibodies against iκB (39 kDa), P65 (65 kDa), P44 (42–44 kDa), P38 (40 kDa), JNK (46–55 kDa) (Cat. No. 9926 T, 8242 T, 4814 T, Cell Signaling Technology, Inc., USA), β-actin (45 kDa), and Lamin-B (67 kDa) (Cat. No. sc-374015, sc-47778, Santa Cruz Biotechnology, Inc., USA), diluted in a 3% bovine serum albumin (BSA) solution at a 1 : 1,000 ratio. Subsequently, they were incubated with the corresponding secondary antibodies. Protein bands were visualized using Super ECL Plus detection reagents (Cat. No. H31500, Tianjin Tiandi Renhe Biotech Co., China) and recorded using a chemiluminescence imaging analysis system (Tanon 5200, YuanPingHao Biotech, China).

The kidney tissues were homogenized and lysed in RIPA buffer (Beyotime, Nantong, China) with protease inhibitors. The cells were lysed in whole-cell lysate buffer with phosphatase and protease inhibitors (Beyotime). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Boston, MA, USA). The membranes were incubated with primary antibodies including COX-2 (Abcam, Cambridge, UK; dilution 1 : 1000), HistoneH3 (Beyotime; dilution 1 : 1000), β-actin (ProteinTech, Chicago, IL, USA; dilution 1 : 3000), GAPDH (Kangchen, Shanghai, China; dilution 1 : 5000), p-AKT(Ser473), p-AKT(Thr308), p-mTOR(Ser2448), mTOR, GRP78, p-eIF2α, ATF4, CHOP, LC3 and p62 (CST, Danvers, MA, USA; 1 : 1000) overnight at 4 °C, followed by a horseradish peroxidase-conjugated IgG secondary antibody (dilution 1 : 3000) for 1 h at room temperature. Photographs were captured and the relative density of blotting was quantified using Image J software (NIH, Bethesda, MD, USA).

ICR mice were divided into three groups by a computerized randomization procedure. Mice in the Control group were maintained at ambient temperature (23 ± 1 °C). Mice in the Heat group were exposed to an ambient temperature of 41 ± 0.5 °C in a specific environmental control smart chamber (HOPE-MED 8150E, Tianjing, China) until a core temperature of (Tc) > 42.7 °C was achieved (Heatstroke onset). The Heat group animals were then transferred to an ambient temperature of 23 ± 1 °C to recover for 12 h^{43 (link)}. Tc was monitored at 15-min intervals using a digital thermometer (ALC-ET06, Shanghai Alcott Biotech Co, Shanghai, China) which was inserted 2 cm into the rectum. Mice in the DEX group were injected intraperitoneally with 25 µg/kg DEX (Selleck; USA) (the Control group was injected with 0.9% saline) immediately after the onset of heatstroke^{21 (link)}. Mice were finally anaesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg) and cerebral cortex tissue was harvested from the mice. The tissues were lysed in RIPA lysis buffer (Beyotime Company, Jiangsu, China) containing a cocktail of phosphatase and protease inhibitors (Roche Diagnostics Corp). The supernatant was collected after centrifugation for subsequent protein detection.

Western blotting was performed using whole-cell lysates of breast cancer cells supplemented with phosphatase and protease inhibitors (Beyotime, Shanghai, China). 20μg proteins were separated by 8–12% SDS-PAGE, transferred onto PVDF membranes (Millipore, Burlington, MA, USA), blocked with 5% BSA (BSA dilution in 1× TBST), and incubated with primary antibodies listed in Table S3 for overnight at 4 °C. Signals from HRP-coupled secondary mouse or rabbit antibodies (Abcam, Cambridge, UK) were generated by Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA).
The TCA precipitation method was performed to analyze secreted proteins in the conditioned medium as previously described [35 (link)]. 0.5–1 mL conditioned medium was collected and trichloroacetic acid (Macklin, China) was added to a final concentration of 10%, then vortexed for 1min and incubated on ice for 24 h. The samples were centrifuged at 13000 rpm for 20 min at 4 °C, and the supernatant was discarded and washed twice using 100% ice-cold acetone. Subsequently, dried at 37 °C, followed by dissolved in 50 μL 1× loading buffer and heated for 10 min at 95 °C. 10 μL proteins were separated by 10% SDS-PAGE and followed as described above.